Gotaq pcr kit

The RNA were extracted from the three viral stocks (AnD133719, SHM172805 and ArD141967) and were used as templates for RT-PCR. Specific primers (Table 2), the M-MLV system (Invitrogen, Carlsbad, CA, USA), and the Go-Taq PCR Kit (Promega, Madison, WI, USA) were used for cDNA synthesis and amplification, according to the manufacturer’s instructions. All primers used to amplify the S and M segments were designed according to RVFV sequences available in GenBank. Accession numbers of all RVFV sequences used to design the primers are presented in Additional file 1: Table S1. The PCR products of the expected sizes were purified directly from the agarose gel using a QIAgen Gel extraction kit and sequenced by Cogenics (Beckman Coulter Genomics, Essex, United Kingdom). Sequencing was performed in both directions, using the original reverse and forward primers as for the amplification.Table 2

List of primers used

Nom	Segment	Position	Sequence	Tm
NSng	S	31–48	TATCATGGATTACTTTCC	48
NSca	S	841–824	CCTTAACCTCTAATCAAC	50
M1F	M	3–22	ACAAAGACCGGTGCAACTTC	53.9
M1R	M	1120–1140	CCAYGCAAAGGGTATGCAAT	53.2
M2F	M	1035–1054	TGAGGACTCTGAATTRCACCT	48.7
M2R	M	2395–2415	TCCAGAGAGTTGAGCCTTGC	53.3
MRV1a	M	3050–3068	CAAATGACTACCAGTCAGC	44.6
MRV2g	M	2262–2292	GGTGGAAGGACTCTGCGA	52.5
M3F	M	2979–2998	CAGTCCTCAGTGAGCYCATA	46.1
M3R	M	3763–3782	TCTCGGTTCTGGRGTGTGAA	52.5

All exons and exon-intron boundaries of candidate genes located within homozygous intervals in P3 and P4 were amplified (GoTaq PCR Kit Promega) according to the manufacturer’s protocol and Sanger sequenced. The TRNT1 mutations identified by WES in P1 and P2 were also confirmed by Sanger sequencing, using primer sequences for TRNT1 exons 3 and 6 (exon 3: Forward ATAGCAGGAGGAGCAGTGAG and reverse GACTGCAGGGTTTATGACGG and exon 6 forward CAGATTTTGCTTGTGATATGCCA and reverse ACCCCATAACCCAAACTTTGTC) All PCR products were sequenced using the Big Dye Terminator Reaction Kit on a 2400GeneAmp® 9700 sequencer. Sequencing data were analysed using the Sequencher v. 5.2.4 software.

Overlapping RT-PCRs were done to recover the complete genome. All primer sequences can be found in the S3 Table. The NS5, envelope and NS5-partial 3’UTR regions were first amplified using flavivirus consensus or West Nile specific primers [1 (link),105 (link),106 (link)], followed by amplification of NS3 region using designed WNV primers. The 5’ non-coding region of the genome was obtained using the 5’RACE kit (Invitrogen, Carlsbad, USA) and a designed consensus primer in the capsid protein for reverse primer. Finally, specific primers were designed according to the first sequences obtained and a second step of RT-PCR was done to obtain the complete genome.
The PCR fragments were obtained using AMV reverse transcription kit (Promega, Madison, USA) for reverse transcription and Go-Taq PCR kit (Promega, Madison, USA) for amplification. The RT conditions were set according to the manufacturer’s instructions, and the PCR conditions were as follows: 5 minutes at 95°C, 40 cycles of 1 minute at 95°C, 1 minute at 53°C, 1 to 4 minutes (according the size of the PCR product) at 72°C, and 10 minutes at 72°C. The PCR products were purified from the agarose gel using the Gel extraction kit (Qiagen) and sequenced by Cogenics (Beckman Coulter Genomics, Essex, UK).

Total RNA was isolated using RNAzol B reagent (TelTest) from 6 to 10 Daphnia following a bacterial RNAi feeding regimen for 10 days. Prior to RNA isolation, for the Daphnia fed on bacteria expressing any of the described dsRNAs, the entire gut was removed from each individual to avoid contamination from the bacteria in the gut that contain the dsRNA. Daphnia were collected in a microcentrifuge tube, rinsed once with 1 ml of PBS, and were homogenized in 0.8 ml of RNAzol B. Total RNA was isolated as per the supplied protocol. cDNA was synthesized using random hexamer primers, 1 μg total RNA, 10–20 units M-MuLV reverse transcriptase, 500 μM dNTPs, 40 units RNase Inhibitor RNasin (Promega) in appropriate reaction buffer. For each PCR reaction, 2 μl (1/10th of total) cDNA was used with 50 pmoles each of the forward and reverse primers designed to amplify phenoloxidase PCR product using the Promega GoTaq PCR kit. The following conditions were used for PCR: 95° C for 5 min (initial denaturation), denaturation at 95° C for 30 s, annealing at 58° C for 30 s, extension at 72° C for 30 s for 27 cycles in order to stay within linear range of amplification. The linear range was determined by varying cycle numbers and performing a densitometric analysis of the amplified product. PCR products were separated on a 1 % agarose gel.

Total RNA was extracted from decidual cells and DSCs using Macherey-Nagel RNA isolation kit according to the manufacturer’s protocol. The concentration of the extracted RNA was determined by absorbance at 260 nm and the purity was estimated via A260/A280 ratio with Nanodrop spectrophotometer ND-1000 (Thermo Scientific). To evaluate the quality of RNA extracted, 1 μg of total RNA were reverse transcribed (Superscript II reverse Transcriptase kit, Invitrogen) and amplified (GoTaq PCR kit, Promega) with primers (custom-made by AIT biotech, Singapore) for the housekeeping gene GAPDH. Human macrophages cells, isolated as previously described by Cao et al. (25 (link)), were used as internal control. The PCR amplified products were separated on 1.5% agarose gel containing gel green along with 100-bp ladder (Fermentas) for visualization. GAPDH expression was used an endogenous reference gene and analyzed in parallel in all samples.

RNA was isolated from two pooled enteroid monolayers using TRIzol (Invitrogen) and the Direct-zol miniprep (Zymo Research) according to manufacturer’s protocol. Genomic DNA was removed with the TURBO DNA free kit (Invitrogen) in presence of SUPERase RNAse inhibitor (Invitrogen). cDNA was made with the Superscript IV first strand synthesis kit (Invitrogen) using oligo(dT)₂₀ and random hexamers. Gene expression was assessed by PCR using the cDNA as template, the primers listed in the supplemental material (Table S5) and the GO Taq PCR kit (Promega). Amplicons were visualized on a 1% agarose gel.